The NKX3.1 transcription factor is an NK family homeodomain protein and a tumor suppressor gene that is haploinsufficient and down-regulated in the early phases of prostate cancer. Like its cardiac homolog, NKX2.5, NKX3.1 acts synergistically with serum response factor (SRF) to activate expression from the smooth muscle gamma-actin (SMGA) gene promoter. Using NMR spectroscopy, three conserved motifs in a construct containing the N-terminal region and homeodomain of NKX3.1 were observed to interact with the MADS box domain of SRF. These motifs interacted both in the absence of DNA and when both proteins were bound to a SMGA promoter DNA sequence. No significant interaction was seen between the homeodomain and SRF MADS box. One of the SRF-interacting regions was the tinman (TN) or engrailed homology-1 motif (EH-1), residues 29-35 (FLIQDIL), which for other NK proteins is the site of interaction with the repressor protein Groucho. A second hydrophobic interacting region was designated the SRF-interacting (SI) motif and included residues 99-105 (LGSYLLD). A third interacting motif was the acidic region adjacent to the SI motif including residues 88-96 (ETLAETEPE). The acidic domain (AD) motif signals also showed strengthening upon the NKX3.1 homeodomain binding to DNA in the absence of SRF, consistent with the acidic region weakly interacting with the homeodomain in the unbound state. The importance of these linear motifs in the transcriptional interaction of NKX3.1 and SRF was demonstrated by targeted mutagenesis of an NKX3.1 expression vector in a SMGA reporter assay. The results implicate the NKX3.1 N-terminal region in regulation of transcriptional activity of this tumor suppressor.